This invention relates to the manipulation of plant gene expression and the production of transgenic plants.
Plant growth and development relies on the integration of developmental and environmental signals. In response to these signals, undifferentiated cells at the meristems are able to develop into either vegetative or reproductive structures. The signaling and regulatory mechanisms underlying the maintenance and the differentiation of meristems are mostly unknown. Two plant hormones, auxin and cytokinin, are known to affect meristematic cell division activities. A common phenomenon termed apical dominance results from the suppression of secondary meristems by inhibitory levels of auxin produced at the actively growing apex. Application of cytokinin at the secondary meristems can, however, release this suppression. It is therefore likely that hormones such as auxin and cytokinin are also involved in the maintenance and the regulation of meristem differentiation.
Mitogen-activated protein kinase (MAPK) pathways have been implicated in transmitting hormonal and environmental signals to the cell nucleus in organisms ranging from yeast to humans. For example, in mammals, the primary responses to hormone, growth, and stress signals are mediated by a conserved signaling cascade consisting of three protein kinases, the mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase kinase kinase (MAPKKK). MAPKKK phosphorylates and activates MAPKK that, in turn, phosphorylates and activates MAPK. The activated MAPK can be translocated into the nucleus where it phosphorylates transcription factors that control gene expression (Herskowitz, Cell 80: 187-197, 1995; Kyriakis et al., J. Biol. Chem. 271: 24313-24316, 1996). Additionally, in some mammalian cells, the activation of the same MAPK pathway can lead to either cell proliferation or differentiation depending on the duration of the activation. Moreover, dual-specificity MAPK phosphatases have recently been identified as specific regulators which act to turn off and attenuate MAPK signal transduction pathways.
In one aspect, the invention features a method for modifying a plant phenotype. The method, in general, includes the steps of: (a) introducing into a plant cell a transgene including DNA encoding a phosphatase domain of a dual-specificity mitogen-activating protein kinase (MAPK) phosphatase (or a phosphatase domain thereof) operably linked to a promoter functional in the plant cell to yield a transformed plant cell; and (b) regenerating a transgenic plant from the transformed plant cell, wherein the phosphatase domain of the dual-specificity MAPK phosphatase is expressed in the cells of the transgenic plant, thereby modifying the phenotype of the transgenic plant. In preferred embodiments, the dual-specificity MAPK phosphatase (or a phosphatase domain thereof) hydrolyzes phosphoserine/threonine and phosphotyrosine residues on a protein substrate. In other preferred embodiments, the dual-specificity MAPK phosphatase (or a phosphatase domain thereof) is a eukaryotic dual-specificity MAPK phosphatase (e.g., an approximately full-length MKP-1 or a polypeptide including approximately amino acids 1-314 of MKP-1). In particular applications, the method is useful for modifying a plant""s phenotype for the production of plants having increased yield; increased flower production; early flowering; increased reproductive capacity; decreased vegetative growth; delayed senescence; decreased sensitivity to auxin; increased seed production; or increased regeneration capacity in vitro.
In related aspects, the invention features a plant (or plant cell, plant tissue, plant organ, or plant part) including a transgene capable of expressing a phosphatase domain of a dual-specificity MAPK phosphatase, wherein the transgene is expressed in the transgenic plant under the control of a promoter that is functional in a plant cell. In preferred embodiments, the dual-specificity MAPK phosphatase hydrolyzes phosphoserine/threonine and phosphotyrosine residues on a protein substrate. In other preferred embodiments, the dual-specificity MAPK phosphatase is a eukaryotic dual-specificity MAPK phosphatase (e.g., an approximately full-length MKP-1 or a polypeptide including approximately amino acids 1-314 of MKP-1).
In related aspects, the invention also features seeds and cells from a plant which includes a transgene capable of expressing a phosphatase domain of a dual-specificity MAPK phosphatase (or a phosphatase domain thereof).
In general, the phosphatase domain used in the methods or transgenic plants of the invention is generally expressed by itself, as a dual-specificity MAPK phosphatase polypeptide or phosphatase domain-containing fragment thereof, or as part of a genetically engineered chimeric polypeptide. Useful dual-specificity MAPK phosphatases include those that inactivate a MAPK pathway cascade; improve yields; increase flower production; promote early flowering; increase reproductive season; decrease vegetative growth; delay senescence; decrease sensitivity to auxin; increase seed production; or increase plant regeneration in vitro. Exemplary phosphatase domains include, without limitation, those that are substantially identical or identical to the phosphatase domains of MKP-1, MKP-2, MKP-3, MKP-4, PAC-1, MSG5, Pmp1, IphP, AtMKP1, AtMKP2, AtMKP3, or AtMKP3. Preferably, the methods and plants of the invention specifically utilize the phosphatase domain of MKP-1 or amino acids 1-314 of MKP-1. In other preferred embodiments, a full-length dual-specificity MAPK phosphatase polypeptide or a phosphatase domain containing fragment thereof that is substantially identical or identical to MKP-1, MKP-2, MKP-3, MKP-4, PAC-1, MSG5, Pmp1, IphP, AtMKP-1, AtMKP-2, AtMKP-3, or AtMKP-4 is utilized.
The DNA encoding the dual-specificity MAPK phosphatase polypeptide or phosphatase domain-containing fragment thereof is, in general, constitutively expressed in the transgenic plant. However, if desired, the domain may be inducibly expressed, or such a domain may be expressed in a cell-specific, tissue-specific, or organ-specific manner.
In other preferred embodiments, the invention features an isolated nucleic acid molecule including a sequence encoding a dual-specificity MAPK phosphatase having at least 40% identity with the amino acid sequence shown in FIG. 7 (SEQ ID NO: 2). In preferred embodiments, the sequence that encodes a dual-specificity MAPK phosphatase includes the amino acid sequence shown in FIG. 7 (SEQ ID NO: 2). Preferred nucleic acid molecules are obtained from cruciferous plants, for example, Arabidopsis thaliana. An exemplary nucleic acid molecule of a cruciferous dual-specificity MAPK phosphatase is shown in FIG. 7 (SEQ ID NO: 1).
In still another aspect, the invention features a plant including an isolated nucleic acid molecule including a sequence (encoding a dual-specificity MAPK phosphatase having at least 40% identity with the amino acid sequence shown in FIG. 7 (SEQ ID NO: 2). In addition, the invention features seeds and cells from such plants, as well as parts of such plants. These plants may be produced according to conventional methods of molecular biology using any crop or ornamental plant, e.g., those plants described herein.
In yet another aspect, the invention features a substantially pure dual-specificity MAPK phosphatase including an amino acid sequence that has at least 40% (and preferably, 50%, 60%, 70%, 80%, or 90%) identity to the amino acid sequence of FIG. 7 (SEQ ID NO: 2) In preferred embodiments, the polypeptide is obtained from a cruciferous species, for example, Arabidopsis thaliana. 
Exemplary plants which are useful in the methods of the invention, as well as for generating the transgenic plants (or plant cells, plant tissues, plant organs, or plant parts) of the invention, include, without limitation, dicots and monocots, such as sugar cane, wheat, rice, maize, sugar beet, barley, manioc, crucifer, mustard, potato, soybean, sorghum, cassava, banana, grape, oats, tomato, millet, coconut, papaya, orange, rye, cabbage, apple, eggplant, watermelon, canola, cotton, carrot, pepper, strawberry, peanut, legume, bean, pea, mango, and sunflower.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 40%, preferably 50%, more preferably 80%, and most preferably 90%, or even 95% sequence identity to a reference sequence (for example, the amino acid sequences of the phosphatase domains or full-length dual-specificity MAPK phosphatases of MKP-1, MKP-2, MKP-3, MKP-4, PAC-1, MSG5, Pmp1, IphP, AtMKP-1, AtMKP-2, AtMKP-3, or AtMKP-4 or to their respective nucleic acid sequences). For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids or greater. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides or greater.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, FastA, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide (for example, a dual-specificity MAPK phosphatase) that has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, polypeptide. A substantially pure polypeptide may be obtained, for example, by extraction from a natural source (for example, a plant cell); by expression of a recombinant nucleic acid encoding the polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By xe2x80x9cspecifically hybridizesxe2x80x9d is meant that a nucleic acid sequence is capable of hybridizing to a DNA sequence at least under low stringency conditions as described herein, and preferably under high stringency conditions, also as described herein.
By xe2x80x9cobtained fromxe2x80x9d is meant isolated from or having the sequence of a naturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic DNA, or combination thereof).
By xe2x80x9cisolated nucleic acid moleculexe2x80x9d is meant a nucleic acid (e.g., DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene. The term therefore includes, for example, a gene or fragment thereof that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant nucleic acid molecule which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a dual-specificity MAPK phosphatase or a phosphatase domain-containing fragment thereof.
By xe2x80x9creporter genexe2x80x9d is meant a gene whose expression may be assayed; such genes include, without limitation, xcex2-glucuronidase (GUS), luciferase (LUC), chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), and xcex2-galactosidase.
By xe2x80x9ca promoter functional in a plant cellxe2x80x9d is meant any minimal sequence sufficient to direct transcription in a plant cell. Included in the invention are promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, stress-, or hormone-inducible elements or chemical inducers) or elements that are capable of cycling gene transcription; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene or engineered into a transgene construct.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9cplant cellxe2x80x9d is meant any self-propagating cell bounded by a semi-permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
By xe2x80x9ccruciferxe2x80x9d is meant any plant that is classified within the Cruciferae family. The Cruciferae include many agricultural crops, including, without limitation, rape (for example, Brassica campestris and Brassica napus), broccoli, cabbage, brussel sprouts, radish, kale, Chinese kale, kohlrabi, cauliflower, turnip, rutabaga, mustard, horseradish, and Arabidopsis.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a nucleic acid sequence (e.g., a recombinant DNA sequence) which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into the nuclear or plastidic genome.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawings will first be described.